Regulation and Substrate Selectivity of Peptide Exporters and Their Functional Significance in Streptococcus pneumoniae

Peptidase-containing ABC transporters (PCATs) are a widely distributed family of transporters which export peptide substrates containing a double-glycine motif (GG peptides). In gram-positive bacteria, PCATs secrete pheromones used for cell-to-cell signaling and antimicrobial peptides called bacteriocins used for interbacterial killing and competition. PCATs recognize GG peptides through their N-terminal signal sequences, but little is known about how PCATs distinguish between different GG peptides. The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) encodes multiple PCATs. Two of these, ComAB and BlpAB, secrete the quorum-sensing pheromones CSP (competence-stimulating peptide) and BlpC, respectively. CSP induces genetic competence, allowing pneumococcus to incorporate extracellular DNA into its genome via homologous recombination to facilitate DNA repair and horizontal gene transfer. BlpC induces the production of the major family of pneumococcal bacteriocins, the Blp bacteriocins (pneumocins). Recently, it was reported that ComAB could cross-secrete the BlpC pheromone, mediating crosstalk from the competence regulatory system (com) to the pneumocin regulatory system (blp). Here, I extend that work to show that BlpAB can also cross-secrete CSP, enabling crosstalk in the blp to com direction. Moreover, the ability of ComAB and BlpAB to share substrates extends to the pneumocins. While nearly all strains produce functional ComAB and encode pneumocins, only 25% produce functional BlpAB. Cross-secretion of CSP and BlpC by ComAB/BlpAB results in complex patterns of com and blp regulation which differ between BlpAB+ and BlpAB− strains. First, BlpAB+ strains can activate competence at lower cell densities and under a greater range of conditions than BlpAB− strains. Second, BlpAB+ strains can secrete pneumocins independently of competence while BlpAB− strains can only secrete pneumocins during brief periods of competence activation. Moreover, differences in timing and duration of transporter expression between ComAB and BlpAB allow BlpAB+ strains to secrete greater amounts of pneumocins than BlpAB− strains. This leads to a pneumocin-mediated competitive advantage for BlpAB+ strains over BlpAB− strains during nasopharyngeal colonization in mice. Therefore, BlpAB+ strains are aggressors which use pneumocins to kill competitors under a wide range of conditions while BlpAB− strains are opportunists which primarily use pneumocins to support competence. The cross-secretion between com- and blp- regulated peptides led me to examine the role of a previously uncharacterized pneumococcal PCAT, RtgAB, in peptide secretion. RtgAB is encoded by the rtg locus next to several GG peptides of unknown function. I determined that rtg is regulated by the RtgR/RtgS system, in which RtgS, a SHP (small hydrophobic peptide)-like pheromone with a distinctive Trp-X-Trp motif, is exported then reimported back into the cell to induce rtg through the Rgg-family transcription regulator RtgR. An active RtgR/S system provides a competitive fitness advantage in a mouse model of nasopharyngeal colonization. Since ComAB and BlpAB share substrates, I investigated the ability of RtgAB to do the same and found that RtgAB and ComAB/BlpAB preferentially secrete different sets of GG peptides. This selectivity is determined by the GG peptides’ signal sequences; ComAB/BlpAB prefers substrates with certain hydrophobic residues at conserved signal sequence positions while RtgAB prefers substrates with a unique motif at the N-terminal end of the signal sequence. These findings illuminate a relatively understudied part of PCAT biology and will help guide future efforts to predict PCAT-substrate pairings. Ultimately, studying PCAT regulation and how they secrete GG peptides will advance our understanding of the many microbial processes dependent on these transporters.PHDMicrobiology & Immunology PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/174523/1/wangchar_1.pd